Mcdb153 medium

Primary keratinocytes were freshly isolated from infant penile skin, which was discarded as waste at the time of circumcision. Cells in passages 2–4 that had not reached terminal differentiation were used for this study, as reported previously [22 (link)]. They were isolated, selected and cultured in serum-free MCDB-153 medium (Sigma St. Louis, MO USA), which was supplemented with amino acids (50 mg/L of L-histidine, 99 mg/L of L-isoleucine, 14 mg/L of L-methionine, 15 mg/L of L-phenylalanine, 10 mg/L of L-tryptophan, and 14 mg/L of L-tyrosine), and growth factors (2 mg/L bovine pituitary extract, 25 µg/L EGF 5 mg/L insulin, and 50 µg/L PGE1). The medium was changed every other day.

Esomeprazole magnesium hydrate, omeprazole, N-acetylcysteine (NAC), thapsigargin (TG), RPMI-1640, MCDB-153 medium, and antibiotics were from Sigma-Aldrich (Madrid, Spain). Fetal bovine serum (FBS) and Hank's balanced salt solution (HBSS) were both from Life Technologies (Madrid, Spain). All compounds except pepstatin A, which was dissolved in 100% ethanol and NAC, which was dissolved in culture media, were dissolved in DMSO and made up with the media so that the final concentration of the vehicle was not >0.04% (v/v).

WM35, a radial growth phase cell line, was provided Dr. Nikolas Haass and the malignant SK-Mel28 cell line was provided by Dr. Brian Gabrielli (both University of Queensland, Australia). WM35 cells were maintained in MCDB 153 medium (Sigma-Aldrich) supplemented with 4% FCS, 20% Leibovitz’s medium (Life Technologies), bovine insulin (5 µL/mL), CaCl₂ (1.68 mM), sodium bicarbonate (7.5% w/v). SK-Mel-28 cells were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, 25 mM HEPES, 10% FCS. Both media contained Penicillin (50 U/mL) + Streptomycin (50 µg/mL) and 0.01% Gentamycin (Life Technologies). The identity of both these cell lines was authenticated using short tandem repeat (STR) profiling by CellBank Australia and tested for mycoplasma using the MycoAlert^TM mycoplasma detection kit (Lonza).

The B16F10 murine melanoma cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Cell culture was performed as described before [40] (link). α-MSH and dexamethasone (DEX) were purchased from Sigma-Aldrich (MO, USA). To detect the effects of DEX and α-MSH on melanogenesis, cells were incubated with 1 µM DEX or 50 nM α-MSH. B16F10 cells at passage numbers between 5 and 7 were used for the experiments.
Normal human epidermal melanocytes were derived from young male adult foreskins (ethnic Han/aged 18–22 years) obtained at circumcision following standard protocols [41] (link). Melanocytes were incubated with MCDB153 medium (Sigma-Aldrich, USA) containing 1 nM choleratoxin (Sigma-Aldrich, USA), 0.1 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, USA), 1.6 nM phorbolesters (Sigma-Aldrich, USA), 5 µg/mL insulin (Sigma-Aldrich, USA) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. To detect the effects of DEX and α-MSH on melanogenesis, cells were incubated with 1 µM DEX or 50 nM α-MSH. Melanocytes at passage numbers between 3 and 6 were used for the experiments. The studies on human material were approved by Nanjing Drum Tower Hospital, Medical Ethics Committee. All participants provided their written informed consent to participate in this study. This consent procedure was approved by the Nanjing Drum Tower Hospital, Medical Ethics Committee.

Trypan blue, DMEM, RPMI, penicillin/streptomycin, trypsin, siRNA against LC3, MELK and p53 were purchased from Life Technology. Fetal calf serum (FCS) was purchased from HyClone. MCDB 153 medium, metformin, staurosporine and dimethylacetamide were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Labrafil M1944 Cs was purchased from Gatte fosse. Annexin V and DAPI were purchased from Roche Applied Science. PLX4032 was purchased from Euromedex. HSP60, p53, HSP90, Actin, Cyclin E, AMPKα1 and AMPKα2 antibodies were purchased from Santa Cruz Biotechnology (TEBU; Le Perray en Yvelines, France). LC3-B, Ⓟ-AMPKα (T172), Ⓟ-mTOR (S2448), PARP, AMPKα total, mTOR total, Ⓟ-ACC (Ser79), ACC total, MELK, CDK1, Ⓟ-CHK2 (Thr68), CHK2 total, CDK2, Ⓟ-P53 (ser15), Caspase 9 and Caspase 3 antibodies were from Cell Signaling Technology (Berverly, MA, USA). Antibodies against Ⓟ-CDC25B (S323), CDC25B total, gamma-H₂AX, and ATM total were purchased from ABCAM. Antibodies against cyclin D1 and pRb were from BD Bioscience (Pont de Claix, France). REDD1 antibody was purchased from Proteintech. TMRE was purchased from Abcam (ab113852-TMRE Mitochondrial Membrane Potential Assay Kit). ATP production was measured using a Malachite Green Phosphate Assay Kit produced by Cayman chemical (n°10009325). MELK adenovirus and Ⓟ-ATM (10H11.E12) antibody were purchased from TEBU.

Human melanoma cell lines WM793B, A2058, A375, Hs294T and mouse melanoma cell line B16F10 were purchased from the American Type Culture Collection. A2058, A375, Hs294T and B16F10 cells were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, California, USA) and 1% penicillin-streptomycin (Invitrogen). WM793B cell line was maintained in MCDB153 medium (Sigma-Aldrich) with 2% fetal bovine serum (Invitrogen). Human melanoma cell lines UACC62 and UACC257 were given from Dr. Schrama in University Hospital Würzburg in Germany in 2016, and were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Invitrogen). All these melanoma cell lines were authenticated by short-tandem repeat fingerprinting by Fourth Military Medical University in 2016, and these cell lines show no mycoplasma contamination. IFN-γ was purchased from R&D Systems (Minneapolis, Minnesota, USA). Liproxstatin-1 (HY-12726), (1S,3R)-RSL3 (HY-100218A), erastin (HY-15763), ferrostatin-1 (HY-100579) were purchased from MedChemExpress (Monmouth Junction, New Jersey, USA). agomiR-21–3p (hsa-miR-21–3p) and antagomiR-21–3p (hsa-miR-21–3p antagomiR) were purchased from RiboBio (Guangzhou, China).